Technetium-99m labeled peptides for imaging

ABSTRACT

This invention relates to radiolabeled peptides and methods for producing such peptides. Specifically, the invention relates to peptides, methods and kits for making such peptides, and methods for using such peptides to image sites in a mammalian body labeled with technetium-99m (Tc-99m) via Tc-99m binding moieties. In particular, the peptide reagents of the invention are covalently linked to a polyvalent linker moiety, so that the polyvalent linker moiety is covalently linked to a multiplicity of the specific-binding peptides, and the Tc-99m binding moieties are covalently linked to a plurality of the specific-binding peptides, the polyvalent linker moiety, or to both the specific-binding peptides and the polyvalent linker moiety.

This is a divisional of application Ser. No. 07/893,981 filed Jun. 5,1992 now U.S. Pat. No. 5,508,020.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to radiodiagnostic reagents and peptides, andmethods for producing labeled radiodiagnostic agents. Specifically, theinvention relates to specific-binding peptides, methods and kits formaking such peptides, and methods for using such peptides to image sitesin a mammalian body labeled with technetium-99m (Tc-99m) via aradiolabel-binding moiety which forms a complex with Tc-99m. Inparticular, the peptide reagents of the invention are covalently linkedto a polyvalent linker moiety, so that the polyvalent linker moiety iscovalently linked to a multiplicity of the specific-binding peptides,and the Tc-99m binding moieties are covalently linked to a plurality ofthe specific-binding peptides, the polyvalent linker moiety, or to boththe specific-binding peptides and the polyvalent linker moiety.

2. Description of the Prior Art

In the field of nuclear medicine, certain pathological conditions arelocalized, or their extent is assessed, by detecting the distribution ofsmall quantities of internally-administered radioactively labeled tracercompounds (called radiotracers or radiopharmaceuticals). Methods fordetecting these radiopharmaceuticals are known generally as imaging orradioimaging methods.

In radioimaging, the radiolabel is a gamma-radiation emittingradionuclide and the radiotracer is located using a gamma-radiationdetecting camera (this process is often referred to as gammascintigraphy). The imaged site is detectable because the radiotracer ischosen either to localize at a pathological site (termed positivecontrast) or, alternatively, the radiotracer is chosen specifically notto localize at such pathological sites (termed negative contrast).

A number of factors must be considered for optimal radioimaging inhumans. To maximize the efficiency of detection, a radionuclide thatemits gamma energy in the 100 to 200 keV range is preferred. To minimizethe absorbed radiation dose to the patient, the physical half-life ofthe radionuclide should be as short as the imaging procedure will allow.To allow for examinations to be performed on any day and at any time ofthe day, it is advantageous to have a source of the radionuclide alwaysavailable at the clinical site.

A variety of radionuclides are known to be useful for radioimaging,including ⁶⁷ Ga, ^(99m) Tc (Tc-99m), ¹¹¹ In, ¹²³ I, ¹²⁵ I, ¹⁶⁹ Yb or ¹⁸⁶Re. Tc-99m is a preferred radionuclide because it emits gamma radiationat 140 keV, it has a physical half-life of 6 hours, and it is readilyavailable on-site using a molybdenum-99/technetium-99m generator.

The sensitivity of imaging methods using radioactively-labeled peptidesis much higher than other radiopharmaceuticals known in the art, sincethe specific binding of the radioactive peptide concentrates theradioactive signal over the area of interest. Small synthetic peptidesthat bind specifically to targets of interest may be advantageously usedas the basis for radiotracers. This is because: 1. they may besynthesized chemically (as opposed to requiring their production in abiological system such as bacteria or mammalian cells, or theirisolation from a biologically-derived substance such as a fragment of aprotein); 2. they are small, hence non-target bound radiotracer israpidly eliminated from the body, thereby reducing background(non-target) radioactivity and allowing good definition of the target;and 3. small peptides may be readily manipulated chemically to optimizetheir affinity for a particular binding site.

Small readily synthesized labeled peptide molecules are preferred asroutinely-used radiopharmaceuticals. There is clearly a need for smallsynthetic labeled peptides that can be directly injected into a patientand will image pathological sites by localizing at such sites. Tc-99mlabeled small synthetic peptides offer clear advantages as radiotracersfor gamma scintigraphy, due to the properties of Tc-99m as aradionuclide for imaging and the utility of specific-binding smallsynthetic peptides as radiotracer molecules.

Radiolabeled peptides have been reported in the prior art.

Ege et al., U.S. Pat. No. 4,832,940 teach radiolabeled peptides forimaging localized T-lymphocytes.

Olexa et al., 1982, European Patent Application No. 823017009 disclose apharmaceutically acceptable radiolabeled peptide selected from FragmentE₁ isolated from cross-linked fibrin, Fragment E₂ isolated fromcross-linked fibrin, and peptides having an amino acid sequenceintermediate between Fragments E₁ and E₂.

Ranby et al., 1988, PCT/US88/02276 disclose a method for detectingfibrin deposits in an animal comprising covalently binding aradiolabeled compound to fibrin.

Hadley et al., 1988, PCT/US88/03318 disclose a method for detecting afibrin-platelet clot in vivo comprising the steps of (a) administeringto a patient a labeled attenuated thrombolytic protein, wherein thelabel is selectively attached to a portion of the thrombolytic proteinother than the fibrin binding domain; and (b) detecting the pattern ofdistribution of the labeled thrombolytic protein in the patient.

Lees et al., 1989, PCT/US89/01854 teach radiolabeled peptides forarterial imaging.

Sobel, 1989, PCT/US89/02656 discloses a method to locate the position ofone or more thrombi in an animal using radiolabeled, enzymaticallyinactive tissue plasminogen activator.

Stuttle, 1990, PCT/GB90/00933 discloses radioactively labeled peptidescontaining from 3 to 10 amino acids comprising the sequencearginine-glycine-aspartic acid (RGD), capable of binding to an RGDbinding site in vivo.

Maraganore et al., 1991, PCT/US90/04642 disclose a radiolabeled thrombusinhibitor comprising (a) a inhibitor moiety; (b) a linker moiety; and(c) and anion binding site moiety.

Rodwell et al., 1991, PCT/US91/03116 disclose conjugates of "molecularrecognition units" with "effector domains".

Tubis et al., 1968, Int. J. Appl. Rad. Isot. 19: 835-840 describelabeling a peptide with technetium-99m.

Sundrehagen, 1983, Int. J. Appl. Rad. Isot. 34: 1003 describes labelingpolypeptides with technetium-99m.

The use of chelating agents for radiolabeling polypeptides, and methodsfor labeling peptides and polypeptides with Tc-99m are known in theprior art and are disclosed in co-pending U.S. patent applications Ser.Nos. 07/653,012, 07/807,062, 07/851,074, and 07/871,282, which arehereby incorporated by reference.

Although optimal for radioimaging, the chemistry of Tc-99m has not beenas thoroughly studied as the chemistry of other elements and for thisreason methods of radiolabeling with technetium are not abundant. Tc-99mis normally obtained as Tc-99m pertechnetate (TcO₄ ; technetium in the+7 oxidation state), usually from a molybdenum-99/technetium-99mgenerator. However, pertechnetate does not bind well to other compounds.Therefore, in order to radiolabel a peptide, Tc-99m pertechnetate mustbe converted to another form. Since technetium does not form a stableion in aqueous solution, it must be held in such solutions in the formof a coordination complex that has sufficient kinetic and thermodynamicstability to prevent decomposition and resulting conversion of Tc-99meither to insoluble technetium dioxide or back to pertechnetate.

For the purpose of radiolabeling, it is particularly advantageous forthe Tc-99m complex to be formed as a chelate in which all of the donorgroups surrounding the technetium ion are provided by a single chelatingligand. This allows the chelated Tc-99m to be covalently bound to apeptide through a single linker between the chelator and the peptide.

These ligands are sometimes referred to as bifunctional chelating agentshaving a chelating portion and a linking portion. Such compounds areknown in the prior art.

Byrne et al., U.S. Pat, No. 4,434,151 describe homocysteinethiolactone-derived bifunctional chelating agents that can coupleradionuclides to terminal amino-containing compounds that are capable oflocalizing in an organ or tissue to be imaged.

Fritzberg, U.S. Pat. No. 4,444,690 describes a series oftechnetium-chelating agents based on 2,3-bis(mercaptoacetamido)propanoate.

Byrne et al., U.S. Pat. Nos. 4,571,430 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclidesthat can couple radionuclides to terminal amino-containing compoundsthat are capable of localizing in an organ or tissue to be imaged.

Byrne et al., U.S. Pat. Nos. 4,575,556 describe novel homocysteinethiolactone bifunctional chelating agents for chelating radionuclidesthat can couple radionuclides to terminal amino-containing compoundsthat are capable of localizing in an organ or tissue to be imaged.

Davison et al., U.S. Pat. No. 4,673,562 describe technetium chelatingcomplexes of bisamido-bisthio-ligands and salts thereof, used primarilyas renal function monitoring agents.

Nicolotti et al., U.S. Pat. No. 4,861,869 describe bifunctional couplingagents useful in forming conjugates with biological molecules such asantibodies.

Fritzberg et al., U.S. Pat. No. 4,965,392 describe various S-protectedmercaptoacetylglycylglycine-based chelators for labeling proteins.

Fritzberg et al., European Patent Application No. 86100360.6 describedithiol, diamino, or diamidocarboxylic acid or amine complexes usefulfor making technetium-labeled imaging agents.

Dean et al., 1989, PCT/US89/02634 describe bifunctional coupling agentsfor radiolabeling proteins and peptides.

Flanagan et al., European Patent Application No. 90306428.5 discloseTc-99m labeling of synthetic peptide fragments via a set of organicchelating molecules.

Albert et al., European Patent Application No. WO 91/01144 discloseradioimaging using radiolabeled peptides related to growth factors,hormones, interferons and cytokines and comprised of a specificrecognition peptide covalently linked to a radionuclide chelating group.

Dean, co-pending U.S. patent application Ser. No. 07/653,012 teachesreagents and methods for preparing peptides comprising a Tc-99mchelating group covalently linked to a specific binding peptide forradioimaging in vivo, and is hereby incorporated by reference.

Baidoo & Lever, 1990, Bioconjugate Chem. 1: 132-137 describe a methodfor labeling biomolecules using a bisamine bisthiol group that gives acationic technetium complex.

It is possible to radiolabel a peptide by simply adding athiol-containing moiety such as cysteine or mercaptoacetic acid. Suchprocedures have been described in the prior art.

Schochat et al., U.S. Pat. No. 5,061,641 disclose direct radiolabelingof proteins comprised of at least one "pendent" sulfhydryl group.

Dean et al., co-pending U.S. patent application Ser. No. 07/807,062teach radiolabeling peptides via attached groups containing free thiols,and is incorporated herein by reference.

Goedemans et al., PCT Application No. WO 89/07456 describe radiolabelingproteins using cyclic thiol compounds, particularly 2-iminothiolane andderivatives.

Thornback et al., EPC Application No. 90402206.8 describe preparationand use of radiolabeled proteins or peptides using thiol-containingcompounds, particularly 2-iminothiolane.

Stuttle, PCT Application No. WO 90/15818 describes Tc-99m labeling ofRGD-containing oligopeptides.

Although it is possible to label specific-binding peptides with Tc-99m(as disclosed in co-pending U.S. patent applications Ser. No.07/053,012, now abandoned, which issued as U.S. Pat. No. 5,654,272; Ser.No. 07/807,026, now U.S. Pat. No. 5,443,815; Ser. No. 07/851,074, nowabandoned, which issued as U.S. Pat. No. 5,711,931; and Ser. No.07/871,282, a divisional of which issued as U.S. Pat. No. 5,720,934, allof which are incorporated by reference), some such peptides exhibit lowbinding site affinity whereby the strength of peptide binding to thetarget site is insufficient to allow enough of the radioisotope tolocalize at the targeted site and form a radioimage. Peptides comprisedof linear arrays of specific binding peptide repeating units have beendescribed in the prior art. However, alternative arrangements ofspecific binding peptide units may be preferable in some cases.

Rodwell et al., 1991, PCT/US91/03116 disclose linear arrays of thepeptide sequence RGD.

The present invention provides reagents comprised of a multiplicity ofspecific-binding peptide moieties, having an affinity for targeted sitesin vivo sufficient to produce a scintigraphically-detectable image. Theincorporation of a multiplicity of specific-binding peptide moieties inthe reagents of the invention permits the use of specific bindingpeptides whose individual binding affinity would not otherwise besufficient to produce a scintigraphically-detectable image in vivo. Inother cases, an improvement in the scintigraphic image produced by aparticular specific-binding peptide is achieved using the reagents ofthis invention.

SUMMARY OF THE INVENTION

The present invention provides reagents useful in preparing radioimagingagents comprising a multiplicity of specific-binding peptide moietiescovalently linked to a polyvalent linker moiety, wherein technetium-99mbinding moieties are covalently linked to the specific-binding peptides,the polyvalent linker moiety, or to both the specific-binding peptidesand the polyvalent linker moieties. The invention also provides Tc-99mlabeled scintigraphic imaging agents prepared from such peptidereagents. The specific-binding peptides of the invention are comprisedof peptides that specifically bind to a target in vivo.

In a first aspect of the present invention, the invention providesreagents comprising a multiplicity of specific binding peptides capableof being Tc-99m labeled for imaging sites within a mammalian body,comprising a specific binding peptide having an amino acid sequence of3-100 amino acids, covalently linked to a polyvalent linking moiety, andTc-99m binding moieties covalently linked to a plurality of thespecific-binding peptides, the polyvalent linker moiety, or both.Preferred embodiments of the invention comprise linear and cyclicspecific binding peptides.

In a second aspect, the present invention provides reagents capable ofbeing Tc-99m labeled for imaging sites within a mammalian body,comprising a multiplicity of specific binding peptide having an aminoacid sequence of 3-100 amino acids, covalently linked to a polyvalentlinking moiety, and a Tc-99m binding moiety covalently linked to aplurality of the specific-binding peptides, the polyvalent linkermoiety, or both, wherein the Tc-99m binding moiety has formula:

    C(pgp).sup.S --(aa)--C(pgp).sup.S

wherein C(pgp)^(S) is a protected cysteine and (aa) is an amino acid. Ina preferred embodiment, the amino acid is glycine. In a preferredembodiment, the peptide comprises between 3 and 30 amino acids.Preferred embodiments of the invention comprise linear and cyclicspecific binding peptides.

In a third embodiment, the invention provides reagents capable of beingTc-99m labeled for imaging sites within a mammalian body, comprising amultiplicity of specific binding peptides having an amino acid sequenceof 3-100 amino acids, covalently linked to a polyvalent linking moiety,and a Tc-99m binding moiety covalenty linked to a plurality of thespecific-binding peptides, the polyvalent linker moiety, or both,wherein the Tc-99m binding moiety comprises a single thiol moiety havingthe formula:

    A.sup.1 --CZ.sup.1 (B.sup.1)--[C(R.sup.1 R.sup.2)].sub.n X.sup.1

wherein

A¹ is H, HOOC, H₂ NOC, or --NHOC;

B¹ is SH or NHR³ ;

X¹ is H, methyl, SH or NHR³ ;

Z¹ is H or methyl;

R¹ and R² are independently H or lower alkyl;

R³ is H, lower alkyl or --C═O;

n is 0, 1 or 2;

and where B¹ is NHR³, X¹ is SH, Z¹ is H and n is 1 or 2; where X¹ isNHR³, B¹ is SH, Z¹ is H and n is 1 or 2; where B¹ is H, A¹ is HOOC, H₂NOC, or --NHOC, X¹ is SH, Z¹ is H and n is 0 or 1; where Z¹ is methyl,X¹ is methyl, A¹ is HOOC, H₂ NOC, or --NHOC,, B¹ is SH and n is 0; andwherein the thiol moiety is in the reduced form. In a preferredembodiment, the peptide comprises between 3 and 30 amino acids.Preferred embodiments of the invention comprise linear and cyclicspecific binding peptides.

In another embodiment, the invention provides peptide reagents capableof being Tc-99m labeled for imaging sites within a mammalian bodycomprising a multiplicity of specific binding peptides having an aminoacid sequence of 3-100 amino acids, covalently linked to a polyvalentlinking moiety, and a Tc-99m binding moiety covalently linked to aplurality of the specific-binding peptides, the polyvalent linkermoiety, or both, wherein the Tc-99m binding moiety has formula: ##STR1##[for purposes of this invention, radiolabel-binding moieties having thisstructure will be referred to as picolinic acid (Pic)-based moieties] or##STR2## wherein X is H or a protecting group and (amino acid) is anyamino acid. For purposes of this invention, radiolabel-binding moietieshaving this structure will be referred to as picolylamine (Pica)-basedmoieties. In a preferred embodiment, the amino acid is glycine and X isan acetamidomethyl protecting group. In additional preferredembodiments, the peptide is comprised between 3 and 30 amino acids.Preferred embodiments of the invention comprise linear and cyclicspecific binding peptides.

Yet another embodiment of the invention provides peptide reagentscapable of being labeled with Tc-99m for imaging sites within amammalian body, comprising a multiplicity of specific binding peptideshaving an amino acid sequence of 3-100 amino acids, covalently linked toa polyvalent linking moiety, and a Tc-99m binding moiety covalentlylinked to the specific-binding peptides, the polyvalent linker moiety,or both, wherein the Tc-99m binding moiety has formula: ##STR3## whereineach R can be independently H, CH₃ or C₂ H₅ ; each (pgp)^(S) can beindependently a thiol protecting group or H; m, n and p areindependently 2 or 3; A² is linear or cyclic lower alkyl, aryl,heterocyclyl, combinations or substituted derivatives thereof; and X ispeptide; and ##STR4## wherein each R is independently H, CH₃ or C₂ H₅ ;m, n and p are independently 2 or 3; A is linear or cyclic lower alkyl,aryl, heterocyclyl, combinations or substituted derivatives thereof; Vis H or CO-peptide; R' is H or peptide; provided that when V is H, R' ispeptide and when R' is H, V is peptide. [For purposes of this invention,radiolabel-binding moieties having these structures will be referred toas "BAT" moieties]. Preferred embodiments of the invention compriselinear and cyclic specific binding peptides.

Specific-binding peptides provided by the invention include but are notlimited to peptides having the following sequences:

(acetyl-F_(D) PRPG)₂ KGGGCamide

(GPRVVERHQSA)₂ KC_(Acm) GC_(Acm) amide

[(GPRP)₂ K]₂ KC_(Acm) GC_(Acm) amide ##STR5## (CC_(Acm) GC_(Acm)GGRGDS)₃ -TSEA (GPRPC_(Acm) GC_(Acm) Camide)₃ -TSEA

(Pic.SC_(Acm) SYNRGDSTCamide)₃ -TSEA

(RALVDTLKGGC_(Acm) GC_(Acm) Camide)₃ -TSEA

(GRGDFC_(Acm) GC_(Acm) Camide)₃ -TSEA

(Pic.GC_(Acm) RALVDTLKFVTQAEGAKCamide)₃ -TSEA

(acetyl-SYNRGDTC_(Acm) GC_(Acm) Camide)₃ -DMAB

The specific binding peptides of the invention are covalently linked toa polyvalent linking moiety. Polyvalent linking moieties of theinvention are comprised of at least 2 identical linker functional groupscapable of covalently bonding to specific binding peptides or Tc-99mbinding moieties. Preferred linker functional groups are primary orsecondary amines, hydroxyl groups, carboxylic acid groups orthiol-reactive groups. In preferred embodiments, the polyvalent linkingmoieties are comprised of a multiplicity of polyvalent linking moietiescovalently linked to form a branched polyvalent linking moiety. Theinvention also comprises complexes of the peptides of the invention withTc-99m and methods for radiolabeling the peptides of the invention withTc-99m. Radiolabeled complexes provided by the invention are formed byreacting the peptides of the invention with Tc-99m in the presence of areducing agent. Preferred reducing agents include but are not limited todithionite ion, stannous ion, and ferrous ion. Complexes of theinvention are also formed by labeling the peptides of the invention withTc-99m by ligand exchange of a prereduced Tc-99m complex as providedherein.

The invention also provides kits for preparing the peptides of theinvention radiolabeled with Tc-99m. Kits for labeling the peptide of theinvention with Tc-99m are comprised of a sealed vial containing apredetermined quantity of a peptide of the invention and a sufficientamount of reducing agent to label the peptide with Tc-99m.

This invention provides methods for preparing peptides of the inventionby chemical synthesis in vitro. In a preferred embodiment, peptides aresynthesized by solid phase peptide synthesis.

This invention provides methods for using Tc-99m labeled peptides forimaging a site within a mammalian body by obtaining in vivo gammascintigraphic images. These methods comprise administering an effectivediagnostic amount of a Tc-99m radiolabeled peptide of the invention anddetecting the gamma radiation emitted by the Tc-99m localized at thesite within the mammalian body.

Specific preferred embodiments of the present invention will becomeevident from the following more detailed description of certainpreferred embodiments and the claims.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides reagents for preparing Tc-99m labeledpeptides for imaging target sites within a mammalian body comprising amultiplicity of specific binding peptide having an amino acid sequenceof 3-100 amino acids, covalently linked to a polyvalent linking moiety,and a Tc-99m binding moiety covalently linked to the specific-bindingpeptides, the polyvalent linker moiety, or both.

Labeling with Tc-99m is an advantage of the present invention becausethe nuclear and radioactive properties of this isotope make it an idealscintigraphic imaging agent. This isotope has a single photon energy of140 keV and a radioactive half-life of about 6 hours, and is readilyavailable from a ⁹⁹ Mo--^(99m) Tc generator. Other radionuclides knownin the prior art have effective half-lives which are much longer (forexample, ¹¹¹ In, which has a half-life of 67.4 h) or are toxic (forexample, ¹²⁵ I).

In the radiolabel binding moieties and peptides covalently linked tosuch moieties that contain a thiol covalently linked to a thiolprotecting groups [(pgp)^(S) ] provided by the invention, thethiol-protecting groups may be the same or different and may be but arenot limited to:

--CH₂ -aryl (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH-(aryl)₂, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--C-(aryl)₃, (aryl is phenyl or alkyl or alkyloxy substituted phenyl);

--CH₂ -(4-methoxyphenyl);

--CH-(4-pyridyl)(phenyl)₂ ;

--C(CH₃)₃ -9-phenylfluorenyl;

--CH₂ NHCOR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --NHCOOR (R is unsubstituted or substituted alkyl or aryl);

--CONHR (R is unsubstituted or substituted alkyl or aryl);

--CH₂ --S--CH₂ -phenyl

Prefereed protecting groups have the formula --CH₂ --NHCOR wherein R isa lower alkyl having 1 and 8 carbon atoms, phenyl or phenyl-substitutedwith lower alkyl, hydroxyl, lower alkoxy, carboxy, or loweralkoxycarbonyl. The most preferred protecting group is anacetamidomethyl group.

Polyvalent linking moieties are covalently linked to the specificpeptides of the invention, the Tc-99m binding moieties, or both.Polyvalent linking moieties provided by the invention are comprised ofat least 2 linker functional groups capable of covalently bonding tospecific binding peptides or Tc-99m binding moieties. Such functionalgroups include but are not limited to primary and secondary amines,hydroxyl groups, carboxylic acid groups and thiol reactive groups.Polyvalent linking moieties are comprised of preferably at least threefunctional groups capable of being covalently linked to specific bindingpeptides or technetium-99m binding moieties. Preferred polyvalentlinking moieties include amino acids such as lysine, homolysine,ornithine, aspartic acid and glutamic acid; linear and cyclic amines andpolyamines; polycarboxylic acids; and activated thiols such as di- andtri-maleimides. Also preferred are embodiments wherein the polyvalentlinking moieties comprise a multiplicity of polyvalent linking moietiescovalently linked to form a branched polyvalent linking moiety. For thepurposes of this invention, the term "branched" polyvalent linkingmoieties is intended to include but are not limited to polyvalentlinking moieties having formula: ##STR6##

Peptides of the present invention can be chemically synthesized invitro. Peptides of the present invention can generally advantageously beprepared on an amino acid synthesizer. The peptides of this inventioncan be synthesized wherein the radiolabel-binding moiety is covalentlylinked to the peptide during chemical synthesis in vitro, usingtechniques well known to those with skill in the art. Such peptidescovalently-linked to the radiolabel-binding moiety during synthesis areadvantageous because specific sites of covalent linkage can bedetermined.

Radiolabel binding moieties of the invention may be introduced into thetarget specific peptide during peptide synthesis. For embodiments [e.g.,Pic-Gly-Cys(protecting group)-] comprising picolinic acid (Pic-), theradiolabel-binding moiety can be synthesized as the last (i.e.,amino-terminal) residue in the synthesis. In addition, the picolinicacid-containing radiolabel-binding moiety may be covalently linked tothe ε-amino group of lysine to give, for example,αN(Fmoc)-Lys-εN[Pic-Gly-Cys(protecting group)], which may beincorporated at any position in the peptide chain. This sequence isparticularly advantageous as it affords an easy mode of incorporationinto the target binding peptide.

Similarly, the picolylamine (Pica)-containing radiolabel-binding moiety[-Cys(protecting group)-Gly-Pica] can be prepared during peptidesynthesis by including the sequence [-Cys(protecting group)-Gly-] at thecarboxyl terminus of the peptide chain. Following cleavage of thepeptide from the resin the carboxyl terminus of the peptide is activatedand coupled to picolylamine. This synthetic route requires that reactiveside-chain functionalities remain masked (protected) and do not reactduring the conjugation of the picolylamine.

Examples of small synthetic peptides containing the Pic-Gly-Cys-chelatorare provided in the Examples hereinbelow. This invention provides forthe incorporation of these chelators into virtually any peptide,resulting in a radiolabeled peptide having Tc-99m held as neutralcomplex.

This invention also provides specific-binding small synthetic peptideswhich incorporate bisamine bisthiol (BAT) chelators which may be labeledwith Tc-99m, resulting in a radiolabeled peptide having Tc-99m held asneutral complex.

In forming a complex of radioactive technetium with the peptides of thisinvention, the technetium complex, preferably a salt of Tc-99mpertechnetate, is reacted with the peptides of this invention in thepresence of a reducing agent. Preferred reducing agents are dithionite,stannous and ferrous ions; the most preferred reducing agent is stannouschloride. In an additional preferred embodiment, the reducing agent is asolid-phase reducing agent. Complexes and means for preparing suchcomplexes are conveniently provided in a kit form comprising a sealedvial containing a predetermined quantity of a peptide of the inventionto be labeled and a sufficient amount of reducing agent to label thepeptide with Tc-99m. Alternatively, the complex may be formed byreacting a peptide of this invention with a preformed labile complex oftechnetium and another compound known as a transfer ligand. This processis known as ligand exchange and is well known to those skilled in theart. The labile complex may be formed using such transfer ligands astartrate, citrate, gluconate or mannitol, for example. Among the Tc-99mpertechnetate salts useful with the present invention are included thealkali metal salts such as the sodium salt, or ammonium salts or loweralkyl ammonium salts.

In a preferred embodiment of the invention, a kit for preparingtechnetium-labeled peptides is provided. The peptides of the inventioncan be chemically synthesized using methods and means well-known tothose with skill in the art and described hereinbelow. Peptides thusprepared are comprised of between 3 and 100 amino acid residues, and arecovalently linked to a radiolabel-binding moiety wherein theradiolabel-binding moiety binds a radioisotope. An appropriate amount ofthe peptide is introduced into a vial containing a reducing agent, suchas stannous chloride or a solid-phase reducing agent, in an amountsufficient to label the peptide with Tc-99m. An appropriate amount of atransfer ligand as described (such as tartrate, citrate, gluconate ormannitol, for example) can also be included. Technetium-labeled peptidesaccording to the present invention can be prepared by the addition of anappropriate amount of Tc-99m or Tc-99m complex into the vials andreaction under conditions described in Example 3 hereinbelow.

Radioactively labeled peptides provided by the present invention areprovided having a suitable amount of radioactivity. In forming Tc-99mradioactive complexes, it is generally preferred to form radioactivecomplexes in solutions containing radioactivity at concentrations offrom about 0.01 millicurie (mCi) to 100 mCi per mL.

Technetium-labeled peptides provided by the present invention can beused for visualizing sites in a mammalian body. In accordance with thisinvention, the technetium-labeled peptides or neutral complexes thereofare administered in a single unit injectable dose. Any of the commoncarriers known to those with skill in the art, such as sterile salinesolution or plasma, can be utilized after radiolabeling for preparingthe injectable solution to diagnostically image various organs, tumorsand the like in accordance with this invention. Generally, the unit doseto be administered has a radioactivity of about 0.01 mCi to about 100mCi, preferably 1 mCi to 20 mCi. The solution to be injected at unitdosage is from about 0.01 mL to about 10 mL. After intravenousadministration, imaging of the organ or tumor in vivo can take place ina matter of a few minutes. However, imaging can take place, if desired,in hours or even longer, after the radiolabeled peptide is injected intoa patient. In most instances, a sufficient amount of the administereddose will accumulate in the area to be imaged within about 0.1 of anhour to permit the taking of scintiphotos. Any conventional method ofscintigraphic imaging for diagnostic purposes can be utilized inaccordance with this invention.

The technetium-labeled peptides and complexes provided by the inventionmay be administered intravenously in any conventional medium forintravenous injection such as an aqueous saline medium, or in bloodplasma medium. Such medium may also contain conventional pharmaceuticaladjunct materials such as, for example, pharmaceutically acceptablesalts to adjust the osmotic pressure, buffers, preservatives and thelike. Among the preferred media are normal saline and plasma.

The methods for making and labeling these compounds are more fullyillustrated in the following Examples. These Examples illustrate certainaspects of the above-described method and advantageous results. TheseExamples are shown by way of illustration and not by way of limitation.

EXAMPLE 1 Synthesis of BAT Chelators

1. Synthesis of TMEA [tris(2-maleimidoethyl)amine]

tris(2-aminoethyl)amine (1.49 mL, 10 mmol) dissolved in 50 mL saturatedaqueous sodium bicarbonate and cooled in an ice bath, was treated withN-carbomethoxymaleimide (4.808 g, 31 mmol). The mixture was stirred for30 min on ice and then for another 30 min at room temperature. Themixture was then partitioned between dichloromethane and water, driedover magnesium sulfate, filtered and evaporated to give 3.442 g ofproduct. Reverse phase thin-layer chromatography (RP-TLC) yieldedessentially 1 spot (R_(f) =0.63 in 1:1 acetonitrile: 0.5 M sodiumchloride). 3.94 mmol (1.817g) of this product was dissolved in 20 mLtetrahydrofuran and 20 mL saturated sodium bicarbonate and mixed for 2h. The reaction mixture was then partitioned between ethyl acetate andwater. The organic phase was washed with saturated sodium chloride,dried over magnesium sulfate, and filtered. The ethyl acetate solutionwas diluted with hexanes and cooled. Solid TMEA was collected byfiltration and dried to a yield of 832 mg. Chemical analysis of theproduct confirmed its identity as TMEA as follows:

¹ H NMR (CDCl₃): 2.65 (tr. 2 H), 3.45 (tr.2 H). 6.64 (s. 2 H).

¹³ C NMR (CDCl₃), 35.5, 51.5, 133.9, 170.4.

2. Synthesis of TMEB(4-[1-(2-tolylsulfonylmethyl)ethenylcarbonyl]benzoic acid)

4-(bis-(2-toluenethiomethyl)acetyl)benzoic acid was prepared from2-thiocresol using the methods of Lawton and co-workers (1990,Bioconjugate Chemistry 1: 36). The identity of the resulting compoundwas established by chemical analysis as follows:

FABMS: MH⁺ =436.

¹ H NMR (CDCl₃)=2.62 (s, 6H), 3.2-3.4 (m, 4H), 3.94 (d tr, 1H),7.10-7.26 (m, 8H), 7.64 (d, 2H), 8.07 (d, 2H).

¹³ C NMR (CDCl₃): 20.2, 34.9, 45.4, 126.5, 126.8, 128.1, 129.9, 130.3,130.4, 132.9, 133.9, 138.9, 140.5.

To a solution of 4-(bis-(2-toluenethiomethyl)acetyl)benzoic acid (1.865g, 4.27 mmol) in 50% methanol/water (12.5 mL) was added acetic acid(2.69 mL) followed by 30% hydrogen peroxide (2.61 mL) and disodiumtungstate dihydrate (0.187 g, 0.56 mmol). The mixture was stirredovernight and the crude product was filtered off. Recrystallization frommethanol/water and reverse-phase HPLC (0.1% CF₃ COOH/acetonitrile/water)gave TMEB (178 mg). The identity of the resulting compound wasestablished by chemical analysis as follows:

¹ H NMR (DMSO-d6): 2.68 (s, 3H), 4.56 (s, 2H), 5.95 (s. 1H), 6.27 (s.1H), 7.37-8.05 (m, 8H).

EXAMPLE 2 Solid Phase Peptide Synthesis

Solid phase peptide synthesis (SPPS) was carried out on a 0.25 millimole(mmole) scale using an Applied Biosystems Model 431A Peptide Synthesizerand using 9-fluorenylmethyloxycarbonyl (Fmoc) amino-terminus protection,coupling with dicyclohexylcarbodiimide/hydroxybenzotriazole or2-(1H-benzo-triazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate/hydroxybenzotriazole (HBTU/HOBT), and usingp-hydroxymethylphenoxymethyl-polystyrene (HMP) resin forcarboxyl-terminus acids or Rink amide resin for carboxyl-terminusamides. Resin-bound products were routinely cleaved using a solutioncomprised of trifluoroacetic acid, water, thioanisole, ethanedithiol,and triethylsilane, prepared in ratios of 100:5:5:2.5:2 for 1.5-3 h atroom temperature.

Where appropriate N-terminal acetyl groups were introduced by treatingthe free N-terminal amino peptide bound to the resin with 20% v/v aceticanhydride in NMP (N-methylpyrrolidinone) for 30 min. Where appropriate2-chloroacetyl or 2-bromoacetyl groups were introduced either by usingthe appropriate 2-haloacetic acid as the last residue to be coupledduring SPPS, or by treating the N-terminus free amino acid peptide boundto the resin with either the 2-haloaceticacid/diisopropylcarbodiimide/N-hydroxysuccinimide in NMP or the2-haloacetic anhydride/diisopropylethylamine in NMP.

Where appropriate, HPLC-purified 2-haloacetylated peptides were cyclizedby stirring an 0.1-1.0 mg/mL solution in phosphate or bicarbonate buffer(pH 8.0) containing 0.5-1.0 mM EDTA for 4-48 h followed by acidificationwith acetic acid, lyophilization and HPLC purification.

Where appropriate, DMAB adducts were prepared by reacting singlethiol-containing peptides (10 to 100 mg/mL in DMF) with 0.5 molarequivalents of TMEB (described in Example 1) and 1 molar equivalent oftriethanolamine at room temperature for approximately 12 to 18 hours.DMF was then removed in vacuo and the product purified by HPLC.

Where appropriate, TSEA adducts were prepared by reacting singlethiol-containing peptides (10 to 100 mg/mL in DMF) with 0.33 molarequivalents of TMEA [tris(2-maleimidoethyl)amine] with or without 1molar equivalent of triethanolamine at room temperature forapproximately 7 hours to 5 days. DMF was then removed in vacuo and theproduct purified by HPLC.

Where appropriate, BSME adducts were prepared by reacting singlethiol-containing peptides (5 to 50 mg/mL in 50 mM sodium phosphatebuffer, pH 8) with 0.5 molar equivalents of BMME(bis-maleimidomethylether) pre-dissolved in acetonitrile at roomtemperature for approximately 1 to 18 hours. The solution wasconcentrated and the product was product by HPLC.

Crude peptides were purified by preparative high pressure liquidchromatography (HPLC) using a Waters Delta Pak C18 column and gradientelution using 0.1% trifluoroacetic acid (TFA) in water modified withacetonitrile. Acetonitrile was evaporated from the eluted fractionswhich were then lyophilized. The identity of each product was confirmedby fast atom bombardment mass spectroscopy (FABMS).

EXAMPLE 3 A General Method for Radiolabeline with Tc-99m

0.1 mg of a peptide prepared as in Example 2 was dissolved in 0.1 mL ofwater or 50 mM potassium phosphate buffer (pH=5, 6 or 7.4). Tc-99mgluceptate was prepared by reconstituting a Glucoscan vial (E.I. DuPontde Nemours, Inc.) with 1.0 mL of Tc-99m sodium pertechnetate containingup to 200 mCi and allowed to stand for 15 minutes at room temperature.25 μl of Tc-99m gluceptate was then added to the peptide and thereaction allowed to proceed at room temperature or at 100° C. for 15-30min and then filtered through a 0.2 μm filter.

The Tc-99m labeled peptide purity was determined by HPLC using theconditions described in the Footnotes in Table I. Radioactive componentswere detected by an in-line radiometric detector linked to anintegrating recorder. Tc-99m gluceptate and Tc-99m sodium pertechnetateelute between 1 and 4 minutes under these conditions, whereas the Tc-99mlabeled peptide eluted after a much greater amount of time.

The following Table illustrates successful Tc-99m labeling of peptidesprepared according to Example 2 using the method described herein.

    __________________________________________________________________________                             FABMS                                                                             Radiochemical                                                                        HPLC                                                               MH.sup.+                                                                          Yield (%)*                                                                           R.sub.T (min)**                           __________________________________________________________________________    (acetyl-F.sub.D PRPG).sub.2 KGGGCamide                                                                 1613                                                                              98.sup.2                                                                             17.4                                      (GPRVVERHQSA).sub.2 KC.sub.Acm GC.sub.Acm amide                                                        2986                                                                              99.sup.3                                                                             16.0                                      [(GPRP).sub.2 K].sub.2 KC.sub.Acm GC.sub.Acm amide                                                     2437                                                                              100.sup.3                                                                            16.3                                       ##STR7##                3021                                                                              ND     ND                                        (CC.sub.Acm GC.sub.Acm GGRGDS).sub.3 -TSEA                                                             ND  82.sup.1                                                                             10.4                                      (GPRPC.sub.Acm GC.sub.Acm Camide).sub.3 -TSEA                                                          3189                                                                              93.sup.1                                                                             10.0                                      (Pic.SC.sub.Acm SYNRGDSTCamide).sub.3 -TSEA                                                            4489                                                                              99.sup.1                                                                             10.4, 11.2                                (RALVDTLKGGC.sub.Acm GC.sub.Acm Camide).sub.3 -TSEA                                                    4998                                                                              95.sup.2                                                                             13.4, 13.7                                (GRGDFC.sub.Acm GC.sub.Acm Camide).sub.3 -TSEA                                                         3561                                                                              ND     ND                                        (Pic.GC.sub.Acm RALVDTLKFVTQAEGAKCamide).sub.3 -TSEA                                                   7244                                                                              98.sup.4                                                                             18.3, 19.0                                (acetyl-SYNRGDTC.sub.Acm GC.sub.Acm Camide).sub.2 -DMAB                                                3087                                                                              ND     ND                                        __________________________________________________________________________     *Superscripts refer to the following labeling conditions:                     .sup.1 The peptide is dissolved in 50 mM potassium phosphate buffer (pH       7.4) and labeled at 100° C.                                            .sup.2 The peptide is dissolved in water and labeled at room temperature.     .sup.3 the peptide is dissolved in a 50:50 mixture comprising 50 mM           potassium phosphate buffer (pH 7.4) and absolute ethanol and labeled at       100° C.                                                                **HPLC methods (indicated by superscript after R.sub.T):                      general:                                                                      solvent A = 0.1% CF3COOH/H.sub.2 O                                            solvent B.sub.90 = 0.1% CF.sub.3 COOH/90% CH.sub.3 CN/H.sub.2 O               solvent flow rate = 1 mL/min                                                  Vydak column = Vydak 218TP54 RP18, 5μ × 220 mm × 4.6 mm        analytical column with guard column                                           Conditions: 100% A to 100% B.sub.90 in 10 min                                 Singleletter abbreviations for amino acids can be found in G. Zubay,          Biochemistry (2d. ed.), 1988 (MacMillen Publishing: New York) p. 33;          Pic = picolinoyl (pyridine2-carbonyl);                                        Acm = acetamidomethyl;                                                        Apc = L[S-(3-aminopropyl)cysteine;                                            F.sub.D = Dphenylalanine;                                                     Y.sub.D = Dtyrosine;                                                          BSME = bissuccinimdylmethylether;                                             DMAB = 4(2,2-dimethylacetyl)benzoic acid;                                     TSEA = tris(succinimidylethyl)amine                                           Peptides are linked to BSME, DMAB or TSEA linkers via the free thiol          moiety of the unprotected cysteine residue (C) in each peptide.          

It should be understood that the foregoing disclosure emphasizes certainspecific embodiments of the invention and that all modifications oralternatives equivalent thereto are within the spirit and scope of theinvention as set forth in the appended claims.

What is claimed is:
 1. A radiolabeled complex comprising:a)technetium-99m; and b) a reagent comprising:i) a multiplicity ofsynthetically prepared, specific-binding peptides, each peptide havingan amino acid sequence of 3 to 100 amino acids, ii) a polyvalent linkercovalently linked to each peptide, and iii) a technetium-99m bindingmoiety covalently linked to a specific site on each peptide.
 2. A methodfor labeling a reagent by ligand exchange, wherein the reagentcomprises:i) a multiplicity of synthetically prepared, specific-bindingpeptides, each peptide having an amino acid sequence of 3 to 100 aminoacids, ii) a polyvalent linker covalently linked to each peeptide, andiii) a technetium-99m binding moiety covalently linked to a specificsite on each peptide;the method comprising the step of combining thereagent with a prereduced technetium-99m complex.
 3. A method forlabeling a reagent comprising:i) a multiplicity of syntheticallyprepared, specific-binding peptides, each peptide having an amino acidsequence of 3 to 100 amino acids, ii) a polyvalent linker covalentlylinked to each peptide, and iii) a technetium-99m binding moietycovalently linked to a specific site on each peptide; the methodcomprising the step of reacting the reagent with technetium-99m in thepresence of a reducing agent.
 4. The method of claim 3, wherein thereducing agent is selected from the group consisting of a dithioniteion, a stannous ion and a ferrous ion.
 5. A radiolabeled complexcomprising technetium-99m and a reagent selected from the groupconsisting of:(acetyl-F_(D) PRPG)₂ KGGGCamide, (GPRVVERHQSA)₂ KC_(Acm)GC_(Acm) amide, {(GPRP)₂ K}₂ KC_(Acm) GC_(Acm) amide, ##STR8## (CC_(Acm)GC_(Acm) GGRGDS)₃ -TSEA, (GPRPC_(Acm) GC_(Acm) Camide)₃ -TSEA,(Pic.SC_(Acm) SYNRGDSTCamide)₃ -TSEA, (RALVDTLKGGC_(Acm) GC_(Acm)Camide)₃ -TSEA, (GRGDFC_(Acm) GC_(Acm) Camide)₃ -TSEA, (Pic.GC_(Acm)RALVDTLKFVTQAEGAKCamide)₃ -TSEA, and (acetyl-SYNRGDTC_(Acm) GC_(Acm)Camide)₂ -DMAB.
 6. A radiolabeled complex comprising:a) technetium-99m;and b) a reagent comprising:(i) a polyvalent linker; (ii) a multiplicityof synthetically prepared, specific-binding peptides, each peptidehaving an amino acid sequence of 3 to 100 amino acids and beingcovalently linked to said linker, and (iii) a technetium-99m bindingmoiety covalently linked to said linker.
 7. A method for labeling areagent by ligand exchange, wherein the reagent comprises:(i) apolyvalent linker; (ii) a multiplicity of synthetically prepared,specific-binding peptides, each peptide having an amino acid sequence of3 to 100 amino acids and being covalently linked to said linker; and(iii) a technetium-99m binding moiety covalently linked to saidlinker;the method comprising the step of combining the reagent with aprereduced technetium-99m complex.
 8. A method for labeling a reagentcomprising:(i) a polyvalent linker; (ii) a multiplicity of syntheticallyprepared, specific-binding peptides, each peptide having an amino acidsequence of 3 to 100 amino acids and being covalently linked to saidlinker, and (iii) a technetium-99m binding moiety covalently linked tosaid linker;the method comprising the step of reacting the reagent withtechnetium-99m in the presence of a reducing agent.
 9. A radiolabeledcomplex comprising:a) technetium-99m; and b) a reagent comprising:i) amultiplicity of synthetically prepared, specific-binding peptides, eachpeptide having an amino acid sequence of 3 to 100 amino acids; ii) abranched polyvalent linker covalently linked to a specific site on eachpeptide; and iii) a technetium-99m binding moiety covalently linked tothe linker.
 10. A method for labeling a reagent by ligand exchange,wherein the reagent comprises:i) a multiplicity of syntheticallyprepared, specific-binding peptides, each peptide having an amino acidsequence of 3 to 100 amino acids; ii) a branched polyvalent linkercovalently linked to a specific site on each peptide; and iii) atechnetium-99m binding moiety covalently linked to the linker,the methodcomprising the step of combining the reagent with a prereducedtechnetium-99m complex.
 11. A method for labeling a reagentcomprising:i) a multiplicity of synthetically prepared, specific-bindingpeptides, each peptide having an amino acid sequence of 3 to 100 aminoacids; ii) a branched polyvalent linker covalently linked to a specificsite on each peptide; iii) a technetium-99m binding moiety covalentlylinked to the linker,the method comprising the step of reacting thereagent with technetium-99m in the presence of a reducing agent.
 12. Aradiolabeled complex comprising:a) technetium-99m; and b) a reagentcomprising:i) a multiplicity of synthetically prepared, specific-bindingpeptides, each peptide having an amino acid sequence of 3 to 100 aminoacids; ii) a technetium-99m binding moiety covalently linked to aspecific site on each peptide; and iii) a polyvalent linker covalentlylinked to each technetium-99m binding moiety.
 13. A method for labelinga reagent by ligand exchange, wherein the reagent comprises:i) amultiplicity of synthetically prepared, specific-binding peptides, eachpeptide having an amino acid sequence of 3 to 100 amino acids; ii) atechnetium-99m binding moiety covalently linked to a specific site oneach peptide; and iii) a polyvalent linker covalently linked to eachtechnetium-99m binding moiety;the method comprising the step ofcombining the reagent with a prereduced technetium-99m complex.
 14. Amethod for labeling a reagent comprising:i) a multiplicity ofsynthetically prepared, specific-binding peptides, each peptide havingan amino acid sequence of 3 to 100 amino acids; ii) a technetium-99mbinding moiety covalently linked to a specific site on each peptide; andiii) a polyvalent linker covalently linked to each technetium-99mbinding moiety;the method comprising the step of reacting the reagentwith technetium-99m in the presence of a reducing agent.
 15. A method ofmaking a technetium-99m labeled peptide comprising the step of combining##STR9## with technetium-99m in the presence of stannous ions.
 16. Amethod of making a technetium-99m labeled peptide by ligand exchangecomprising the step of combining ##STR10## with a prereducedtechnetium-99m complex.
 17. The complex of any of claims 1, 6, 9 or 12,wherein the linker comprises at least two identical functional groups.18. The complex of claim 17, wherein the functional groups are selectedfrom the group consisting of primary amines, secondary amines, hydroxylgroups, carboxylic acid groups, and thiol-reactive groups.
 19. A complexaccording to any of claims 1, 6, or 12, wherein the linkers form acovalently linked, branched polyvalent linking moiety.
 20. The complexof any of claims 1, 6, 9, or 12, wherein the technetium-99m bindingmoiety is selected from the group consisting of:

    C(pgp).sup.S --(aa)--C(pgp).sup.S                          I.

wherein c(pgp)^(S) is a cysteine having a protected thiol group and (aa)is an amino acid; a technetium-99m binding moiety comprising a singlethiol having a formula

    A.sup.1 --CZ.sup.1 (B.sup.1)--[C(R.sup.1 R.sup.2)].sub.n --X.sup.1II.

wherein A¹ is H, HOOC, H₂ NOC, or --NHOC; B¹ is SH or NHR³ ; X¹ is H,methyl, SH or NHR³ ; Z¹ is H or methyl; R¹ and R² are independently H orlower alkyl; R³ is H, lower alkyl or --C═O; n is 0, 1 or 2;and where B¹is NHR³, X¹ is SH, Z¹ is H and n is 1 or 2; where X¹ is NHR³, B¹ is SH,Z¹ is H and n is 1 or 2; where B¹ is H, A¹ is HOOC, H₂ NOC, or --NHOC,X¹ is SH, Z¹ is H and n is 0 or 1; where Z¹ is methyl, X¹ is methyl, A¹is HOOC, H₂ NOC, or --NHOC, B¹ is SH and n is 0; and wherein the thiolmoiety is in the reduced form; ##STR11## wherein X² ═H or a protectinggroup; (amino acid)═any amino acid; ##STR12## wherein X² ═H or aprotecting group; (amino acid)═any amino acid; ##STR13## wherein each Ris independently H, CH₃ or C₂ H₅ ; each (pgp)^(S) is independently athiol protecting group or H; m, n and p are independently 2 or 3; A²=linear or cyclic lower alkyl, aryl, heterocyclyl, or a combinationthereof;and ##STR14## wherein each R is independently H, CH₃ or C₂ H₅ ;m, n and p are independently 2 or 3; A³ =linear or cyclic lower alkyl,aryl, heterocyclyl, or a combination thereof; V═H or --CO-peptide; R⁴ ═Hor peptide;and wherein when V═H, R⁴ ═peptide and when R⁴ ═H,V═--CO-peptide.
 21. The complex of any of claims 1, 6, 9, or 12,whereinthe technetium-99m binding moiety is selected from the group consistingof a protected thiol-containing moiety, a single thiol containingcomplexing moiety, a picolinic acid-based moiety, apicolylamine-containing moiety, and a bisamine bisthiol-containingmoiety.
 22. The complex of any of claims 1, 6, 9, or 12,wherein thelinker comprises an amino acid selected from the group consisting oflysine, homocysteine, ornithine, aspartic acid, and glutamic acid.